Anti-glypican-1-immunizing antigen receptor

ABSTRACT

The purpose of the present invention is to produce a chimeric antigen receptor (CAR) specific to glypican-1 (GPC-1) and to treat squamous cell carcinoma with genetically modified cells capable of expressing the CAR. The present invention provides: a chimeric antigen receptor for use in the treatment and/or prevention of squamous cell carcinoma, said chimeric antigen receptor comprising an extracellular domain capable of binding to GPC-1, a transmembrane domain and one or multiple intracellular domains, wherein at least one of the intracellular domains is an intracellular domain containing a primary cytosolic signaling sequence or an intracellular domain containing both a primary cytosolic signaling sequence and a secondary cytosolic signaling sequence; a genetically modified cell capable of expressing the chimeric antigen receptor; and a cell preparation containing the cell.

The present application is a National Stage Application under 35 U.S.C.§ 371 of International Application No. PCT/JP2016/068924 filed Jun. 24,2016, which claims the benefit of priority to Japanese PatentApplication No. 2015-127001 filed Jun. 24, 2015, the disclosures of allof which are hereby incorporated by reference in their entireties. TheInternational Application was published in Japanese on Dec. 29, 2016 asWO 2016/208754.

TECHNICAL FIELD

The present invention relates to a chimeric antigen receptor (CAR)specific to glypican-1 (GPC-1) and nucleic acid encoding the same, togenetically modified cells that express the CAR, and to a method andcell preparation for treatment and/or prevention of squamous cellcarcinoma using the cells.

BACKGROUND

Squamous cell carcinoma is a commonly appearing form of cancer, and isknown to be a highly invasive and metastatic cancer. Squamous cellcarcinoma has a relatively high relapse rate and a considerably highmortality rate. While squamous cell carcinoma can be diagnosed bybiopsy, it is typically not as distinct as basal cell carcinoma ormelanoma, and its detection and diagnosis are difficult. Theconventional treatment methods, namely surgery, radiation therapy andchemotherapy, require continuous monitoring because of the metastaticnature of the disease. The development of different detection methodsand treatment methods is therefore desired.

Research has been progressing in recent years with the aim of treatingprogressive stage cancer by inducing T cells to recognize cancer cells.Antibody treatment is also being conducted using antibodies formolecules specifically expressed by cancer cells, but therapy byantibodies alone requires frequent administration, while the cytotoxiceffect on cancer cells is also known to be relatively weak.

Treatment methods also exist that use CAR-T cells, prepared by creatingantibodies for molecules specifically expressed by cancer cells andtransferring the genes for the antibody variable regions into T cells(NPL 1). Anti-CD19-CAR-T cells have already been shown to exhibit adramatic clinical effect, mainly against lymphatic leukemia (PTL 1).However, CAR-T cells have only actually exhibited an adequate clinicaleffect for treatment of lymphatic leukemia when using theanti-CD19-CAR-T cells mentioned above. No reports exist ofgenetically-modified T cells for CAR-T cell therapy against solidtumors.

CITATION LIST Patent Literature

-   [PTL 1] Japanese Patent Public Inspection No. 2015-509716

Non-Patent Literature

-   [NPL 1] Nakazawa, S, “Genetically-modified T cell therapy using    chimeric antigen receptor (CAR)”, Shinshu Ishi, 61(4):197-203, 2013

SUMMARY OF INVENTION Technical Problem

In light of the problems of the prior art described above, it is anobject of the present invention to provide a nucleic acid sequence thatcan be used in a method of CAR-T therapy for solid tumors such assquamous cell carcinoma, genetically-modified T cells containing thenucleic acid sequence, and a method of treatment and/or prevention ofsquamous cell carcinoma.

Solution to Problem

The present inventors have previously found that glypican-1 (GPC-1) isspecifically expressed in squamous cell carcinomas such as esophagealcancer, lung cancer and cervical cancer, which are solid tumors, andhave created an anti-GPC-1 antibody (WO2015/098112). In addition, wehave succeeded in using the gene for the anti-GPC-1 antibody to createanti-GPC-1-CAR-T cells, finding that the CAR-T cells exhibit very highGPC-1-specific cytotoxicity against solid tumors, and have thereuponcompleted this invention.

Specifically, the present invention provides the following.

[1] Nucleic acid encoding a chimeric antigen receptor comprising anextracellular domain capable of binding to glypican-1 (GPC-1), atransmembrane domain and one or more intracellular domains, wherein atleast one of the intracellular domains is an intracellular domaincontaining a primary cytosolic signaling sequence.

[2] Nucleic acid according to [1] above, wherein the extracellulardomain capable of binding to GPC-1 includes the heavy chain variableregion (VH) and light chain variable region (VL) of anti-GPC-1 antibody.

[3] Nucleic acid according to [2] above, wherein the nucleotide sequenceencoding the heavy chain variable region of anti-GPC-1 antibody includesthe nucleotide sequence listed as SEQ ID NO: 1 or a nucleotide sequencewith at least 95% identity therewith and having the same function, andthe nucleotide sequence encoding the light chain variable regionincludes the nucleotide sequence listed as SEQ ID NO: 2 or a nucleotidesequence with at least 95% identity therewith and having the samefunction.

[4] Nucleic acid according to any one of [1] to [3] above, wherein theprimary cytosolic signaling sequence contains an immunoreceptortyrosine-based activation motif (ITMA).

[5] Nucleic acid according to [4] above, wherein the intracellulardomain containing the ITAM is derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ,CD3ε, CD5, CD22, CD79a, CD79b or CD66d.

[6] Nucleic acid according to any one of [1] to [5] above, wherein thechimeric antigen receptor further includes one or more identical ordiffering intracellular domains containing a secondary cytosolicsignaling sequence.

[7] Nucleic acid according to [6] above, wherein the intracellulardomain containing the secondary cytosolic signaling sequence is locatedat the N-terminal end of the intracellular domain containing the primarycytosolic signaling sequence.

[8] Nucleic acid according to [6] or [7] above, wherein theintracellular domain containing the secondary cytosolic signalingsequence is derived from CD2, CD4, CD5, CD8α, CD8β, CD28, CD134, CD137,ICOS and/or CD154.

[9] A chimeric antigen receptor encoded by nucleic acid according to anyone of [1] to [8] above.

[10] A vector containing nucleic acid according to any one of [1] to [8]above.

[11] Cells expressing a chimeric antigen receptor, having a genetransferred using a vector according to [10] above.

[12] Cells according to [11] above, wherein the cells are T cells or acell population including T cells.

[13] A cell preparation containing cells according to [12] above, fortreatment and/or prevention of a solid tumor expressing GPC-1.

[14] A cell preparation according to [13] above, wherein the solid tumoris squamous cell carcinoma.

[15] A medical composition comprising nucleic acid according to any oneof [1] to [8] above, a chimeric antigen receptor according to [9], avector according to [10] or cells according to [11] or [12], and amedically acceptable excipient.

[16] A medical composition according to [15] above, for treatment and/orprevention of a solid tumor expressing GPC-1.

[17] A medical composition according to [16] above, wherein the solidtumor is squamous cell carcinoma.

[18] The use of nucleic acid according to any one of [1] to [8] above, achimeric antigen receptor according to [9], a vector according to [10]or cells according to [11] or [12], for production of a drug fortreatment and/or prevention of a solid tumor expressing GPC-1.

[19] The use according to [18] above, wherein the solid tumor issquamous cell carcinoma.

[20] A method for treatment and/or prevention of a solid tumorexpressing GPC-1, wherein nucleic acid according to any one of [1] to[8] above, a chimeric antigen receptor according to [9], a vectoraccording to [10], cells according to [11] or [12], a cell preparationaccording to [13] or [14] or a medical composition according to any oneof [15] to [17] is administered to an individual in need of treatment.

[21] The method according to [20] above, wherein the solid tumor issquamous cell carcinoma.

Advantageous Effects of Invention

According to the invention there is provided a chimeric antigen receptoruseful in the field of adoptive immunogene therapy targeting GPC-1antigen for squamous cell carcinoma, nucleic acid encoding the chimericantigen receptor, and cells expressing the chimeric antigen receptor.Cells having the transferred chimeric antigen receptor of the inventionexhibit high specificity and cytotoxicity against squamous cellcarcinoma cells.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the structure of a vector incorporating an immunogenreceptor. LS: CD8a chain-derived lead sequence (SEQ ID NO: 5), Linker:(Gly4Ser)-3 linker, CD28: human CD28-derived, CD3ζ-ICD: human-derivedCD3ζ intracellular domain (including the stop codon).

FIG. 2 shows expression of an immunogen receptor (GPC-1-CAR) in createdGPC-1-CAR-T cells.

FIG. 3 shows GPC-1-specific IFN-γ and TNF-α production by immunogenreceptor-expressing T cells.

FIG. 4 shows GPC-1-specific IL4 and IL5 production by immunogenreceptor-expressing T cells.

FIG. 5 shows GPC-1-specific cytolysis by immunogen receptor-expressing Tcells.

FIG. 6 shows GPC-1-specific IFN-γ production by immunogenreceptor-expressing T cells, and GPC-1-specific cytolysis by the Tcells.

FIG. 7 shows in vivo suppression of tumor cell increase in mice with atransplanted human esophageal cancer cell line (TE14), by immunogenreceptor-expressing T cells.

FIG. 8 shows in vivo suppression of tumor cell increase in micetransplanted with a forced GPC-1 expression mouse colon cancer cell line(MC38), by immunogen receptor-expressing T cells.

DESCRIPTION OF EMBODIMENTS

The present invention provides chimeric antigen receptor (CAR)-T cellsfor treatment of squamous cell carcinoma specifically expressingglypican-1 (GPC-1), and relates to a method of immunotherapy using thecells. The term “chimeric antigen receptor T cells” (hereunder alsoreferred to simply as “CAR-T cells”) means T cells expressing a chimericantigen receptor (CAR). CAR is a general term encompassing chimericproteins having, for example, a single-chain antibody with the variableregion heavy chain (VH) and light chain (VL) of a tumor antigen-specificmonoclonal antibody (scFv) bonded at the N-terminal end, and a T cellreceptor (TCR)ζ chain, at the C-terminus. T cells expressing CAR, afterhaving recognized a tumor antigen in the scFv region, transmit therecognition signal into the T cells via the ζ chain. A costimulatorydomain may also be inserted between the scFv and ζ chain of an immunogenreceptor for augmented activation of the T cells.

As used throughout the present specification, the term “single-chainantibody (scFv)” means a single-stranded polypeptide derived from anantibody, that retains the ability to bind antigen. An example is anantibody polypeptide having the Fv regions of the immunoglobulin heavychain (H chain) and light chain (L chain) fragments linked via a spacersequence, the polypeptide having been formed by recombinant DNAtechnology. Various methods for creating scFv are known, examplesincluding the methods described in U.S. Pat. No. 4,694,778; Science,242, 423-442(1988) and Nature, 334, 54454(1989).

As used throughout the present specification, the term “domain” means aregion within a polypeptide that is folded into a specific structureindependent from the other regions. Throughout the presentspecification, “domain” is used in the terms “extracellular domain”,“transmembrane domain” and “intracellular domain”, depending on itslocation in the chimeric antigen receptor molecule.

(1) Chimeric Antigen Receptor (CAR) of the Invention

The CAR of the invention comprises (i) an extracellular domain capableof binding to glypican-1 (GPC-1), (ii) a transmembrane domain and (c) atleast one intracellular domain, in that order from the N-terminal end.The CAR of the invention has a high level of expression in cells, andcells expressing the CAR of the invention have a high cell growth rateand high cytokine production levels, and are highly specific for andcytotoxic against cells with CAR-binding GPC-1 antigen on the surface.

(a) Extracellular Domain

The “extracellular domain capable of binding to glypican-1 (GPC-1)” usedin the CAR of the invention is a domain containing an oligo- orpolypeptide that can bind to GPC-1 antigen as the target, and ittypically includes the antigen-binding domain of anti-GPC-1 antibody.The domain, upon binding to and interacting with GPC-1 antigen, such asGPC-1 antigen localized on cancer cell surfaces, imparts specificity tothe CAR-expressing cells. According to the invention, a particularlyuseful extracellular domain to be used is that of an antibody (heavychain (H chain) and light chain (L chain)), and especially the domainthat binds the antigen, such as the antibody Fab fragment and theantibody variable region (heavy chain variable region (VH) and lightchain variable region (VL)). It is most preferred to use scFv. For scFv,the VH and VL may be directly linked in any desired order, or they maybe indirectly linked via a spacer. The amino acid sequence and chainlength of a spacer to be used for linkage of the VH and VL is notrestricted, and any one may be selected with modification asappropriate. According to one specific mode, the extracellular domain tobe used for the invention preferably has scFv comprising a heavy chainvariable region having the amino acid sequence listed as SEQ ID NO: 3and a light chain variable region having the amino acid sequence listedas SEQ ID NO: 4, in any desired order.

The extracellular domain of the CAR of the invention has the property ofbinding to GPC-1 antigen, and as mentioned above, the extracellulardomain is preferably the scFv portion of anti-GPC-1 antibody. Theanti-GPC-1 antibody source of the scFv to be used for the invention maybe the anti-GPC-1 antibody previously created by the present inventors(PCT/JP2014/006455), or a monoclonal anti-GPC-1 antibody newly createdwith GPC-1 as antigen, using publicly known technology.

According to one mode, the extracellular domain to be used for theinvention may also have another extracellular domain linked eitherdirectly or indirectly via a spacer to the extracellular domain havingthe aforementioned property of binding to GPC-1 antigen, at itsC-terminal end. The other extracellular domain used may be theextracellular domain of a costimulatory molecule, as described below.

(b) Transmembrane Domain

The CAR of the invention comprises a transmembrane domain. Thetransmembrane domain may be derived from a natural polypeptide, or itmay be one that has been artificially designed. A transmembrane domainderived from a natural polypeptide may be any desired membrane-bound ortransmembrane protein (of a costimulatory molecule, for example). Asused in the present specification, the term “costimulatory molecule”means a cognate binding partner on T cells, that specifically binds to acostimulatory ligand on the target cell membrane, thereby mediating acostimulatory response by T cells, such as cell proliferation, cytolyticactivity or cytokine secretion. Examples of typical costimulatorymolecules that may be used include the transmembrane domains of CD2,CD4, CD5, CD8α, CD8β, CD28, CD134, CD137, ICOS and CD154. Anartificially designed transmembrane domain is a polypeptide consistingmainly of hydrophobic residues such as leucine and valine. Preferably, aphenylalanine-tryptophan-valine triplet is present on each end of asynthetic transmembrane domain. In some cases, a short oligopeptidelinker or polypeptide linker, such as a linker that is a sequence with alength of 2 to 10 amino acids, may be situated between the transmembranedomain and the intracellular domain described below.

As one mode of the invention, the transmembrane domain used may be atransmembrane domain having a sequence from CD28 (for example, aminoacid Nos. 153 to 179 of NCBI RefSeq: NP_006130.1).

The CAR of the invention may have a spacer domain situated between theextracellular domain and the transmembrane domain, or between theintracellular domain and the transmembrane domain. A spacer domain isany oligopeptide or polypeptide that performs the role of linking atransmembrane domain and an extracellular domain and/or a transmembranedomain and an intracellular domain. A spacer domain contains up to 300amino acids, preferably 10 to 100 amino acids and most preferably 25 to50 amino acids.

(c) Intracellular Domain

The intracellular domain to be used for the invention is a moleculecapable of transferring a signal into the cell when an extracellulardomain within the same molecule has bound to (interacted with) antigen.One of the features of the CAR of the invention is that it includes theCD3ζ intracellular domain as the intracellular domain. CD3 is atransmembrane polypeptide that associates with T cell receptor (TCR) andforms a TCR-CD3 complex. CD3 has γ, δ, ε and ζ chains as polypeptides,and forms a heterodimer or homodimer. The nucleotide sequences and aminoacid sequences of all of the polypeptides are known. According to theinvention, therefore, data relating to the nucleotide sequence of theCD3ζ intracellular domain can be obtained by searching for CD3 cDNAsequences using a commonly used nucleotide sequence database.

The CD3ζ intracellular domain may also include mutant sequences havingthe same function. Here, the term “mutant” means any mutant thatincludes a deletion, substitution or addition of one, several or moreamino acids, the mutant preferably retaining the same function as thewild type.

The signal produced through a TCR complex alone is usually insufficientfor activation of T cells, and a secondary signal (costimulatory signal)is often required. Natural T cell activation is transmitted by twodifferent cytosolic signaling sequences, namely a sequence thatinitiates antigen-dependent primary activation via the TCR complex(primary cytosolic signaling sequence), and a sequence actingindependently of the antigen and providing a secondary or costimulatorysignal (secondary cytosolic signaling sequence). According to apreferred mode, the CAR of the invention includes a primary cytosolicsignaling sequence and/or a secondary cytosolic signaling sequence asintracellular domains.

The primary cytosolic signaling sequence regulates primary activation ofthe TCR complex. A primary cytosolic signaling sequence that stimulatesactivation sometimes includes a signaling motif known as immunoreceptortyrosine-based activation motif. (ITAM) (see Nature, 338, 383-384,1989). A primary cytosolic signaling sequence that functions in aninhibitory manner, on the other hand, includes a signaling motif knownas immunoreceptor tyrosine-based inhibitory motif (ITIM) (see JImmunol., 162, 897-902, 1999). According to the invention, anintracellular domain with ITAM, or ITIM depending on the case, may beused.

Intracellular domains with ITAM that may be used for the invention arenot restricted, and include ITAM derived from CD3ζ, FcRγ, FcRβ, CD3γ,CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d. Specifically, there maybe mentioned peptides having the sequences comprising amino acid Nos. 51to 164 of CD3ζ (NCBI RefSeq: NP_932170.1), amino acid Nos. 45 to 86 ofFcεRIγ (NCBI RefSeq: NP 004097.1), amino acid Nos. 201 to 244 of FcεRIβ(NCBI RefSeq: NP_000130.1), amino acid Nos. 139 to 182 of CD3γ (NCBIRefSeq: NP_000064.1), amino acid Nos. 128 to 171 of CD3δ (NCBI RefSeq:NP_000723.1), amino acid Nos. 153 to 207 of CD3ε (NCBI RefSeq:NP_000724.1), amino acid Nos. 402 to 495 of CD5 (NCBI RefSeq:NP_055022.2), amino acid Nos. 707 to 847 of CD22 (NCBI RefSeq:NP_001762.2), amino acid Nos. 166 to 226 of CD79a (NCBI RefSeq:NP_001774.1), amino acid Nos. 182 to 229 of CD79b (NCBI RefSeq: NP000617.1), and amino acid Nos. 177 to 252 of CD66d (NCBI RefSeq:NP_001806.2), as well as mutants thereof having the same function. Theseamino acid numbers, based on the NCBI RefSeq ID or GenBank amino acidsequence data listed in the present specification, are numbers assignedassuming the respective protein precursors (including the signal peptidesequence) as the full length. When the specific molecules mentionedabove are used as the intracellular domain, the intracellular domainand/or transmembrane domain in the molecule can be appropriatelyutilized in the chimeric antigen receptor of the invention.

For this embodiment, the chimeric antigen receptor of the invention mayinclude, in addition to the intracellular domain containing the primarycytosolic signaling sequence, also one or more identical or differingintracellular domains, containing a secondary cytosolic signalingsequence. There are no particular restrictions on intracellular domainscontaining secondary cytosolic signaling sequences that may be used forthe invention, and they include sequences derived from CD2, CD4, CD5,CD8α, CD8β, CD28, CD134, CD137, ICOS and CD154. Specifically, there maybe mentioned peptides having the sequences comprising amino acid Nos.236 to 351 of CD2 (NCBI RefSeq: NP_001758.2), amino acid Nos. 421 to 458of CD4 (NCBI RefSeq: NP_000607.1), amino acid Nos. 402 to 495 of CD5(NCBI RefSeq: NP_055022.2), amino acid Nos. 207 to 235 of CD8α (NCBIRefSeq: NP_001759.3), amino acid Nos. 196 to 210 of CD8β(GenBank:AAA35664.1), amino acid Nos. 181 to 220 of CD28 (NCBI RefSeq:NP 006130.1), amino acid Nos. 214 to 255 of CD137 (4-1BB, NCBI RefSeq:NP_001552.2), amino acid Nos. 241 to 277 of CD134 (OX40, NCBI RefSeq:NP_003318.1) and amino acid Nos. 166 to 199 of ICOS (NCBI RefSeq:NP_036224.1), as well as mutants thereof having the same function. Whenthe specific molecules mentioned above are used as the intracellulardomain, the intracellular domain and/or transmembrane domain in themolecule can be appropriately utilized in the chimeric antigen receptorof the invention.

According to one specific embodiment, the chimeric antigen receptor(CAR) of the invention comprises, from the N-terminus to the C-terminus,(i) an extracellular domain containing the heavy chain variable regionand light chain variable region (or the light chain variable region andheavy chain variable region) of anti-GPC-1 antibody, (ii) anintracellular domain, transmembrane domain and/or intracellular domainselected from among CD2, CD4, CD5, CD8α, CD8β, CD28, CD134, CD137, ICOS,CD154 and their combinations, and (iii) an intracellular domain selectedfrom among CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79band CD66d. According to a preferred embodiment, the CAR of the inventioncomprises, from the N-terminus to the C-terminus, (i) an extracellulardomain comprising the heavy chain variable region (SEQ ID NO: 3) andlight chain variable region (SEQ ID NO: 4) (or the light chain variableregion and heavy chain variable region) of anti-GPC-1 antibody, (ii) theintracellular domain, transmembrane domain and intracellular domain ofCD28, and (iii) the intracellular domain of CD3ζ (see Example 1). Theheavy chain and light chain variable regions may also include mutants ofthese having the same function. Here, the term “mutant” means any mutantincluding a deletion, substitution or addition of one, several or moreamino acids, the mutant preferably retaining the same function as thewild type.

CAR comprising multiple intracellular domains may have oligopeptidelinkers or polypeptide linkers inserted between the intracellulardomains to link them. The linkers used preferably have lengths composedof 2 to 10 amino acids. For example, a linker with a continuous Gly-Sersequence may be used.

(2) Nucleic Acid Encoding Chimeric Antigen Receptor (CAR) of theInvention

According to the invention there is provided nucleic acid encoding theamino acid sequence of CAR according to (1) above. Unless otherwisespecified, the term “nucleic acid (or nucleotide sequence) encoding anamino acid sequence” encompasses all nucleotide sequences that aremutually degenerate forms and code for the same amino acid sequence. Solong as the nucleotide sequence encoding the protein can include anintron in some form, the term “nucleotide sequence” encoding protein andRNA may also include introns.

The nucleic acid encoding the CAR can be easily created by a commonmethod for specified CAR amino acid sequences. A nucleotide sequenceencoding the amino acid sequence of each domain mentioned above can beobtained from the NCBI RefSeq ID or GenBank Accession No. for the aminoacid sequence, and nucleic acid of the invention can be prepared using astandard molecular biological and/or chemical procedure. For example,nucleic acid can be synthesized based on the nucleotide sequences, orDNA fragments obtained from a cDNA library using Polymerase ChainReaction (PCR) may be combined to create the nucleic acid of theinvention. According to a particular mode, the nucleic acid encoding theextracellular domain to be used in the nucleic acid of the invention ispreferably a nucleotide sequence encoding the heavy chain variableregion of anti-GPC-1 antibody (SEQ ID NO: 1) and a nucleotide sequenceencoding the light chain variable region (SEQ ID NO: 2). The nucleotidesequences encoding the heavy chain and light chain variable regions mayalso be substantially homologous nucleotide sequences having the samefunction. The term “substantially homologous” includes two or morebiological molecular sequences that are significantly analogous on theprimary structural nucleotide sequence level. For example,“substantially homologous”, in the context of two or more nucleic acidsequences, means at least about 75% identity, preferably at least about80% identity, more preferably at least about 85% identity or at leastabout 90% identity, and yet more preferably at least about 95% identity,even yet more preferably at least about 97% identity, still yet morepreferably at least about 98% identity, and most preferably at leastabout 99% identity.

The nucleic acid of the invention may be linked with another nucleicacid so as to be expressed under the control of a suitable promoter. Thepromoter used may be one that promotes constitutive expression, or oneinduced by a drug or the like (for example, tetracycline ordoxorubicin). In order to achieve efficient transcription of the nucleicacid, it may be linked with nucleic acid containing other regulatingelements such as an enhancer sequence or terminator sequence thatfunction in combination with the promoter or transcription initiationsite. In addition to the nucleic acid of the invention, a gene that canserve as a marker for confirmation of expression of the nucleic acid(for example, a drug resistance gene, a gene coding for a reporterenzyme, or a gene coding for a fluorescent protein) may also be used inappropriate combinations. An example of a suitable promoter is theimmediate-early cytomegalovirus (CMV) promoter sequence. This promotersequence is a powerful constitutive promoter sequence that can drivehigh-level expression of a desired polynucleotide sequence that isfunctionally linked to it. The term “functionally linked” as used hererefers to functional linkage between a regulatory sequence and aheterogenous nucleic acid sequence that results in expression of thelatter. For example, when a first nucleic acid sequence is situated in afunctional relationship with a second nucleic acid sequence, the firstnucleic acid sequence is said to be functionally linked with the secondnucleic acid sequence. For example, if a promoter affects transcriptionor expression of a coding sequence, the promoter is “functionallylinked” with the coding sequence. Functionally linked DNA sequences areusually contiguous, and are in the same reading frame when it isnecessary to join two protein coding regions.

Other examples of suitable promoters that may be used include ElongationFactor-1α (EF-1α), simian virus 40 (SV40) early promoter, mouse mammarytumor virus (MMTV), human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter,Epstein-Barr virus immediate early promoter, Raus sarcoma viruspromoter, and human gene promoters including, but not limited to, actinpromoter, myosin promoter, hemoglobin promoter and creatine kinasepromoter, although there is no limitation to these, and otherconstitutive promoter sequences may be used. Moreover, the presentinvention is not restricted to the use of constitutive promoters.Inducible promoters are also implied as components of the presentinvention. The use of an inducible promoter provides a molecular switchthat can switch on expression of a polynucleotide sequence to which itis functionally linked, when such expression is desired, or switch itoff when such expression is not desired. Examples of inducible promotersinclude, but are not limited to, metallothionein promoter,glucocorticoid promoter, progesterone promoter and tetracyclinepromoter.

(3) Method for Producing Cells Expressing Chimeric Antigen Receptor(CAR) of the Invention

The method for producing cells expressing the CAR of the inventioncomprises a step of transferring nucleic acid encoding CAR according to(2) above into cells. The step is carried out ex vivo. For example, itcan be produced by transformation of cells ex vivo utilizing a virusvector or a non-virus vector that includes the nucleic acid of theinvention.

The method of the invention may employ cells from a mammal, such ashuman-derived cells or cells derived from a non-human mammal such as amonkey, mouse, rat, pig, cow or dog. The cells to be used in the methodof the invention are not particularly restricted, and any desired cellsmay be used. For example, cells harvested, isolated, purified andinduced from body fluid, tissue or organs, such as blood (peripheralblood or umbilical cord blood) or bone marrow may be used. Peripheralblood mononuclear cells (PBMC), immunocytes (for example, dendriticcells, B cells, hematopoietic stem cells, macrophages, monocytes, NKcell or hematocytes (neutrophils or basophils)), umbilical cord bloodmononuclear cells, fibroblasts, adipocyte precursors, hepatocytes, skinkeratinocytes, mesenchymal stem cells, adipose stem cells, variouscancer cell lines and neural stem cells may be used. According to theinvention it is preferred to use T cells, T cell precursors(hematopoietic stem cells, lymphocyte precursors and the like) or cellpopulations containing them. T cells include CD8⁺ T cells, CD4⁺ T cells,regulatory T cells, cytotoxic T cells and tumor-infiltratinglymphocytes. Cell populations containing T cells and/or T cellprecursors include PBMC. The cells may be ones harvested from the body,or further subjected to amplifying culturing, or ones established ascell lines. When it is desirable to transplant the producedCAR-expressing cells or cells differentiated to such cells into thebody, it is preferred to transfer the nucleic acid into cells harvestedfrom the body itself or a body of the same species.

According to the invention, nucleic acid encoding the CAR of theinvention may be inserted into a vector and the vector introduced intocells. For example, retrovirus vectors (including oncoretrovirusvectors, lentivirus vectors and pseudotyped vectors) or virus vectorssuch as adenovirus vectors, adeno associated virus (AAV) vector, simianvirus vector, vaccinia virus vector or Sendai virus vector, Epstein-Barrvirus (EBV) vector and HSV vector, may be used. Preferred virus vectorsare those incapable of replication so that they cannot auto-replicate inthe infected cells.

Non-virus vectors may also be used for the invention, in combinationwith liposomes, or condensation agents such as the cationic lipidsdescribed in WO96/10038, WO97/18185, WO97/25329, WO97/30170 andWO97/31934. The nucleic acid of the invention can be introduced into thecells by calcium phosphate transfection, lipofection, DEAE-dextran,electroporation or particle bombardment.

When a retrovirus vector is used, for example, appropriate packagingcells may be selected based on the LTR sequence and packaging signalsequence of the vector, and the cells used to prepare retrovirusparticles. Examples of packaging cells include PG13 (ATCC CRL-10686),PA317 (ATCC CRL-9078), GP+E-86 or GP+envAm-12 (U.S. Pat. No. 5,278,056),and Psi-Crip (Proc. Natl. Acad. Sci. USA, Vol. 85, p. 6460-6464(1988)).In addition, retrovirus particles can be formed using 293 cells or 293Tcells that have high transfection efficiency. Retrovirus vectorsproduced based on numerous types of retroviruses, and packaging cellsthat can be used for packaging of the vectors, are widely available fromvarious manufacturers.

A functional substance that increases transfer efficiency may be used inthe step of transferring the nucleic acid into cells (WO95/26200 andWO00/01836, for example). Substances that increase transfer efficiencyinclude substances with activity of binding to virus vectors, such asfibronectin and fibronectin fragments. It is preferred to use afibronectin fragment with a heparin-binding site, such as the fragmentmarketed as RetroNectin CH-296 (Takara Bio, Inc.). Another substancethat may be used is polybrene, as a synthetic polycation having theeffect of increasing infection efficiency of the retrovirus into cells,or fibroblast growth factor, type V collagen, polylysine orDEAE-dextran.

(4) Cells Expressing Chimeric Antigen Receptor (CAR) of the Invention

The cells expressing the CAR of the invention are cells in which nucleicacid encoding the CAR of (2) above has been transferred and expressed bythe production method of (3) above.

The cells of the invention are activated upon transfer of the signalinto the cells in response to binding with a specific antigen throughCAR. Activation of the CAR-expressing cells will differ depending on thetype of host cells and/or the intracellular domain of the CAR, and forexample, release of cytokines, augmentation of cell proliferation orcell surface molecule modification may be used as an index forconfirmation. For example, release of a cytotoxic cytokine (tumornecrosis factor, lymphotoxin or the like) from the activated cellscauses disruption of the target cells expressing the antigen(specifically, squamous cell carcinoma cells). In addition, otherimmunocytes such as B cells dendritic cells, NK cells or macrophages arestimulated by cytokine release or cell surface molecule modification.

(5) Cell Preparation and Medical Composition of the Invention, andMethod of Treatment and Prevention Using them

According to the invention, cells expressing the chimeric antigenreceptor (CAR) can be used for treatment and/or prevention of a disease,and typically a cell preparation and medical composition may can beprovided. The term “treatment” as used here includes mitigation(alleviation) of characteristic symptoms or accessory symptoms of atarget disease, and arrest or retardation of aggravation of symptoms,and treatment also includes improvement in the disease. The term“prevention” means halting or delaying onset or expression of a disease(disorder) or its symptoms, or reducing the risk of its onset orexpression. According to one mode, the cell preparation of the inventionmay comprise CAR-expressing cells of the invention as an activeingredient, together with an appropriate excipient. According to anothermode, the medical composition of the invention comprises nucleic acid,CAR, vector and/or cells of the invention in an effective amount as anactive ingredient, together with an appropriate medically acceptableexcipient. The excipient to be included in the cell preparation andmedical composition may be any of various cell culture media,phosphate-buffered saline, isotonic brine, or the like. The diseaseswhich may be targets of treatment by CAR-expressing cells include solidtumors specifically expressing GPC-1, and more specifically pancreaticcarcinoma, breast cancer, brain tumors and various squamous cellcarcinomas. The squamous cell carcinomas are not particularly restrictedand include esophageal cancer, lung cancer and cervical cancer. Theindividual with a disease to be treated is not particularly restricted,and may be a mammal such as a primate, human, dog, cat, cow, horse, pigor sheep, but it is preferably a human. For treatment of the disease, atherapeutically effective amount of the cell preparation of theinvention is administered to a patient. The term “effective amount” asused in the present specification means an amount that provides atherapeutic or prophylactic advantage. The route of administration isnot restricted, as is recognized by those skilled in the art, and may beparenteral administration, such as intracutaneous, intramuscular,subcutaneous, intraperitoneal, intranasal, intraarterial, intravenous,intratumoral or afferent vessel administration, by injection orinfusion.

The following examples serve as illustration of different modes of thepresent disclosure. It will be apparent to a person skilled in the artthat various modifications may be made to both the materials and methodswhile still being within the scope of the present disclosure. All of thereagents and solvents purchased from commercial product suppliers wereused without further purification or processing.

EXAMPLES Example 1: Construction of Anti-GPC-1-CAR Gene-Carrying VirusVector

The nucleotide sequences coding for the heavy chain variable region (VH)and light chain variable region of anti-GPC-1 antibody were identifiedfrom nucleotide sequence data for the heavy chain and light chainportions of monoclonal anti-GPC-1 antibody (WO2015/098112), that wereprepared from chicken at the National Institutes of BiomedicalInnovation, Health and Nutrition, one of the applicants. Kozaksequence-LS (leader sequence)-VL-linker-VH sequence (Type A) or-LS-VH-linker-VL sequence (Type B) double-stranded DNA was synthesizedand cloned in a CAR expression vector (pMS3-F) (FIG. 1). The recombinantretrovirus vector was transfected into G3T-hi cells together with pGPvector and pE-Eco vector, to prepare a retrovirus vector solution forinfection. PG13 cells (GaLV env.) were infected with the solution tocreate producer cells. An anti-GPC-1-CAR gene-carrying retrovirus vectorsolution (two types, Type A and Type B) were prepared from the cells.

Example 2: Preparation of Anti-GPC-1-CAR-T Cells

Blood was collected from a human and the peripheral blood mononuclearcells (PBMC) were separated using Lymphoprep (#1114544 by Axis-Shield).The separated PBMCs were added to AIM-V (Life Technologies #087-0112DK)+10% human AB serum (Gemini #100-512) to a cell concentration of 2×10⁶/2ml/well. Human rIL2 (500 IU/ml) and anti-human CD3 antibody (OKT-3) (50ng/ml) were added, and after seeding in a 24-well plate, culturing wascarried out at 37° C., 5% CO₂ for 2 days. On the following day, 10 μLRetroNectin (#T100B by Takara Bio, Inc.) (1 mg/ml)+400 uL BS was addedto each well of a separate non-treated 24-well plate (BD #351147), andthe plate was allowed to stand overnight at 4° C. On the following day,the RetroNectin-coated plate was rinsed with 1 ml of PBS, and thendeveloped with 3% BSA/PBS at 500 uL/well and allowed to stand for 30minutes at room temperature, and then rinsed with 1 ml of PBS. Theretrovirus vector solution (Type A or Type B) carrying theanti-GPC-1-CAR gene prepared in Example 1 was diluted 2- to 5-fold, andadded to the RetroNectin-coated plate at 1 ml/well. The plate wascentrifuged at 3044 rpm, 32° C. for 2 hours to adsorb the virus on theRetroNectin at the bottom of the plate. After centrifugation, the virussolution was removed, the PBMCs that had been previously cultured for 2days were added at 5×10⁵/500 μL (AIM-V+10% human AB serum)/well, andhuman rIL2 (500 IU/ml) was added. After centrifugation at 2153 rpm for10 minutes, culturing was initiated at 37° C., 5% CO₂. On the followingday, 1.5 ml of AIM-V+10% human AB serum and human rIL2 (500 IU/ml) wereadded. The number of wells was doubled thereafter each time the cellsreached confluency.

Example 3: IFN-γ Production Test

Human activated peripheral blood mononuclear cells were infected withthe GPC-1-CAR gene-carrying retrovirus vector (Type A or Type B), andthen anti-human CD8 antibody, anti-human CD4 antibody or anti-chickenIgY antibody was used for staining to confirm expression of GPC-1-CAR(FIG. 2). A cell sorter was then used to separate the CD8-positiveanti-GPC-1-CAR-T cells and CD4-positive anti-GPC-1-CAR-T cells. The Tcells (1-2×10⁵ cells) and a GPC-1 forced expression cell line(LK-GPC1(G11)) or a GPC-1 non-expressing cell line (LK-MOCK) (1×10⁵cells) were suspended in 200 μl of AIM-V+10% human AB serum and seededin a 96-well plate. After 24 hours, the culture supernatant wascollected and the IFN-γ, TNF-α, IL-4 and IL-5 concentrations weremeasured by ELISA. Production of IFN-γ, TNF-α, IL-4 and IL-5 by thegenetically modified T cells was measured as a high level ofGPC-1-specific expression (see FIG. 3 and FIG. 4).

Example 4: Cytotoxicity Test

After labeling a GPC-1 forced expression cell line (LK-GPC1(G11)) or aGPC-1 non-expressing cell line (LK-MOCK) with Calcein-AM, as the targetcells, 5×10³ cells were seeded in a 96-well plate. CD8-positiveanti-GPC-1-CAR-T cells or CD4-positive anti-GPC-1-CAR-T cells were addedas effector cells in 40-fold, 20-fold, 10-fold, 5-fold and 2.5-foldamounts, and after 4 hours of culturing, the Calcein-AM in thesupernatant was measured with a fluorophotometer and the proportion ofcells injured by the T cells was calculated. The anti-GPC-1-CAR-T cellslysed the GPC-1-specific cells at a very high rate compared to the Mock(FIG. 5).

Example 5: Recognition of Esophageal Cancer Cells and Toxicity Effect byAnti-GPC-1-CAR-T Cells

The test methods described in Examples 3 and 4, were used to examinerecognition of and toxicity effect on GPC-1-expressing esophageal cancercell lines (“TE8” and “TE14”) by anti-GPC-1-CAR-T cells. As testexamples in addition to the esophageal cancer cell line, there were useda GPC-1 forced expression cell line (“LK2-GPC1(G11)” and“LK2-GPC1(G52)”), a GPC-1-non-expressing cell line (“LK-MOCK (E4)”) anda system without addition of anti-GPC-1-CAR-T cells (“no stimulator”).As shown in FIG. 6A, the anti-GPC-1-CAR-T cells produced IFN-γ inresponse to the GPC-1-expressing cells (“TE8”, “TE14”, “LK2-GPC1(G11)”and “LK-GPC1(G52)”). On the other hand, IFN-γ production by theanti-GPC-1-CAR-T cells was not observed with “LK2-MOCK (E4)” and “nostimulator” that were used as negative controls. Likewise, no IFN-γproduction was observed with control T cells and “no T cells” (nostimulation by T cells), which where not anti-GPC-1-CAR-T cells. Thisdemonstrates that induction of IFN-γ production by anti-GPC-1-CAR-Tcells is GPC-1-specific.

A cytotoxicity test was conducted by the same method as Example 4, using“TE8” and “LK2-MOCK” as the test cells. With GPC-1-expressing TE8, thecytolysis rate of TE8 increased as the proportion of anti-GPC-1-CAR-Tcells added increased, and therefore the anti-GPC-1-CAR-T cells lysedthe cells in a GPC-1-specific manner (FIG. 6B). No cytolysis wasobserved when the “control T cells” were used as the added cells withthe GPC-1-non-expressing cell line (“LK-MOCK)” (FIG. 6C).

Example 6: Treatment Model with Human Esophageal Cancer Cell Line

The human esophageal cancer cell line TE14 (3×10⁶ cells) wassubcutaneously transplanted into NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug/Jic)mice (groups). After 9 days (with confirmation that the TE14 graft hadtaken), anti-GPC-1-CAR-T cells or activated T cells without CAR genetransfer (negative control group) were intraperitoneally administered at2.5×10⁷ cells each. The tumor volume (mm³) in each mouse wasperiodically measured, calculating the long diameter×shortdiameter×short diameter/2 value, and the results are shown FIG. 7. Thenegative control group was a system administerednon-CAR-gene-transferred activated T cells, and the tumor volume wasshown to increase with time (FIG. 7A). On the other hand, in a systemadministered anti-GPC-1-CAR-T cells, where “aGPC-1-CAR-T_1” and“aGPC-1-CAR-T_2” respectively correspond to “GPC-1-CAR_L-H” and“GPC-1-CAR_H-L” shown in FIG. 2, it is seen that increase in tumorvolume was suppressed when using these cells (FIGS. 7A and 7B). FIG. 7Dshows the change in tumor volume of each as the mean±SD. In all of thesystems with addition of anti-GPC-1-CAR-T cells, marked suppression oftumor volume increase is seen compared to the negative control group.

Example 7: Treatment Model with GPC-1 Forced Expression Mouse ColonCancer Cell Line

Since the GPC-1 CAR gene of the invention can recognize both human andmouse GPC-1, mouse cells were used for the subsequent treatmentexperiments. Gene transfer of mouse GPC-1 into the mouse colon cancercell line MC38 was carried out using lentivirus vector, to create aforced expression line (MC38-GPC-1). The plasmid shown in FIG. 1 wasused to create an ecotropic retrovirus vector for the GPC-1-CAR gene,and mouse GPC-1-CAR-T cells were prepared. Specifically, first mousespleen cells (2×10⁶) were cultured for 24 hours in complete RPMI 1640medium (RPMI 1640 basal medium with addition of 10% FCS, 10 mM HEPES, 1mM sodium pyruvate, 1% MEM NEAA (Non-essential amino acids) (#11140-050by Gibco), 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol, as finalconcentrations) containing ConA (2 μg/ml), mouse IL-7 (1 ng/ml) andhuman IL-2 (500 IU/ml), to prepare activated mouse T cells. Next, theretrovirus was used to transfer the GPC-1-CAR gene (“GPC-1-CAR_L-H” ofFIG. 2) into the activated mouse T cells, to create GPC-1-CAR-T cells.The transfer method was according to the procedure described in Example2. However, the number of activated mouse T cells added to each well ofthe RetroNectin-coated plate was 2×10⁶. After infection, the cells werecultured in complete RPMI 1640 medium containing human IL-2 (500 IU/mL),anti-mouse CD3 antibody (1 μg/mL) and anti-mouse CD28 antibody (1μg/mL), and the same procedure was carried out again on the followingday. Next, the prepared CAR-T cells were cultured in complete RPMI 1640medium containing human IL-2 (500 IU/mL), and cultured for 5-7 dayswhile doubling the number of wells each time the cells reach confluency.The transfer efficiency of the GPC-1-CAR gene was approximately 20%.Ne×t, 5×10⁵ MC38-GPC-1 cells were subcutaneously transplanted intoC57BL/6 mice, which were exposed to 5 Gy of radiation after 3 days(after confirming that the graft had taken). The previously preparedGPC-1-CAR-T cells (2×10⁷) were then intraperitoneally administered.Beginning on the same day, IL2 was intraperitoneally administered twicea day for 3 consecutive days (50,000 IU/mouse/dose).Non-CAR-gene-transferred activated T cells were administered to thenegative control group. The tumor volume (mm³) in each mouse wasmeasured, calculating the long diameter×short diameter×short diameter/2value. The results of periodically recording the change in tumor volumein each group are shown in FIG. 8. FIGS. 8A and 8B show changes in tumorvolume for each mouse individual in the negative control group andGPC-1-CAR-T cell-administered group, and FIG. 8C shows the change intumor volume for each group as the mean±SD. As clearly seen from theresults in FIG. 8C, the tumor volume increase was markedly suppressed inthe mice administered the GPC-1-CAR-T cells, compared to the controlgroup. Moreover, there were absolutely no side-effects in the normalmice, and an antitumor effect was observed only in the cancer animalmodel. Incidentally, because these models are in vivo models withnormally functioning immune systems, it may be assumed that theyadequately render the clinical setting for humans. This type ofexperimental system cannot be constructed with other CRT-T cells.

INDUSTRIAL APPLICABILITY

The chimeric antigen receptor of the invention and genetically modifiedcells expressing them are useful for treatment and/or prevention ofsolid tumors such as squamous cell carcinoma, without side-effects.

It is to be noted that other alternative methods exist for carrying outthe embodiments disclosed in the present specification. Therefore, theembodiments are merely for illustrative purposes and should not beconsidered to be restrictive. Moreover, the claims are not restricted bythe detailed description provided in the present specification, and theright of patent applies to the entirety of their scope and equivalentsubject matter.

The invention claimed is:
 1. A nucleic acid encoding a chimeric antigenreceptor comprising an extracellular domain capable of binding toglypican-1 (GPC-1), a transmembrane domain and one or more intracellulardomains, wherein at least one of the intracellular domains is anintracellular domain containing to comprise a primary cytosolicsignaling sequence; wherein the extracellular domain capable of bindingto GPC-1 comprises the heavy chain variable region (VH) and light chainvariable region (VL) of anti-GPC-1 antibody and wherein the nucleotidesequence encoding the heavy chain variable region of anti-GPC-1 antibodycomprises the nucleotide sequence listed as SEQ ID NO: 1 or a nucleotidesequence with at least 95% identity therewith and having the samefunction, and the nucleotide sequence encoding the light chain variableregion comprises the nucleotide sequence listed as SEQ ID NO: 2 or anucleotide sequence with at least 95% identity therewith and having thesame function.
 2. The nucleic acid of claim 1, wherein the primarycytosolic signaling sequence comprises an immunoreceptor tyrosine-basedactivation motif (ITAM).
 3. The nucleic acid of claim 2, wherein theintracellular domain comprising the ITAM is derived from CD3ζ, FcRγ,FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b or CD66d.
 4. The nucleicacid of claim 1, wherein the chimeric antigen receptor further comprisesone or more identical or differing intracellular domains comprising asecondary cytosolic signaling sequence.
 5. The nucleic acid of claim 4,wherein the intracellular domain comprising the secondary cytosolicsignaling sequence is located at the N-terminal end of the intracellulardomain comprising the primary cytosolic signaling sequence.
 6. Thenucleic acid of claim 4, wherein the intracellular domain comprising thesecondary cytosolic signaling sequence is derived from CD2, CD4, CDS,CD8a, CD8β, CD28, CD134, CD137, ICOS and/or CD154.
 7. A chimeric antigenreceptor encoded by the nucleic acid of claim
 1. 8. A vector comprisingthe nucleic acid of claim
 1. 9. Cells comprising the vector of claim 8.10. The cells of claim 9, wherein the cells are T cells or a cellpopulation comprising T cells.
 11. A medical composition comprising thenucleic acid of claim 1, and a medically acceptable excipient.
 12. Anucleic acid encoding a chimeric antigen receptor comprising anextracellular domain capable of binding to glypican-1 (GPC-1), atransmembrane domain and one or more intracellular domains, wherein atleast one of the intracellular domains is an intracellular domaincontaining to comprise a primary cytosolic signaling sequence fortreatment of a solid tumor expressing GPC-1 and wherein the solid tumoris squamous cell carcinoma; wherein the extra cellular domain capable ofbinding to GPC-1 comprises the heavy chain variable region (VH) andlight chain variable region (VL) of anti-GPC-1 antibody and wherein thenucleotide sequence encoding the heavy chain variable region ofanti-GPC-1 antibody and wherein the anti-GPC-I antibody comprises thenucleotide sequence listed as SEQ ID NO: 1 or a nucleotide sequence withat least 95% identity therewith and having the same function, and thenucleotide sequence encoding the light chain variable region comprisesthe nucleotide sequence listed as SEQ ID NO: 2 or a nucleotide sequencewith at least 95% identity therewith and having the same function.